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Additional information

Gene mutation in bacteria

Mutagenicity in bacteria was assessed in a GLP-study performed according to OECD 471 (Notox 2012) with sorbitan monolaurate, ethoxylated (CAS 9005-64-5). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli  WP2uvrA were tested. A plate incorporation assay served as range-finder for the determination of the test concentrations and was considered as a pre-test for toxicity. In the main tests, bacteria were exposed to concentrations of 33, 100, 333, 1000, 3330 and 5000 µg/plate in the plate incorporation assay in the absence and presence of metabolic activation by 5 or 10% rat liver S9-mix. Cytotoxicity was observed in strains TA1535 and TA100 at 3330 µg/plate and above and in TA1537 at 5000 µg/plate without metabolic activation. Further, with metabolic acitvation, cytotoxicity was observed in TA1535 at 5000 µg/plate, in TA1537 at 3330 µg/plate and above and in TA100 at 333, 1000, 3330, 5000 µg/plate. Precipitation of the test substance was not observed. The maximum numbers of revertants observed in the test substance-treated plates were comparable to those in the negative controls for all strains tested, irrespective of metabolic activation. Appropriate positive controls were included into the study design, which gave the expected results and validated the study.

Cytogenicity in mammalian cells

The clastogenic potential of the test substance in-vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD 473 under GLP-conditions (Notox 2012). The selection of the concentrations used for the main study was based on the results of a pre-tests. Based on the findings of cytotoxicity and precipitation, peripheral human lymphocytes were exposed for 3 h to 10, 33 and 100 µg/mL with and without metabolic activation and for 24 h without S9 mix at concentrations of 10, 100 and 300 µg/mL. The harvest time was 24 h after start of exposure. Additionally, cells were exposed for 48 h to 10, 100 and 300 µg/mL without S9 mix and for 3 h to 10, 33 and 100 µg/mL with S9-mix and harvested after 48 h. Cytotoxicity was observed at 300 µg/mL in the continuous experiment without metabolic activation. There were no biologically and statistically significant increases in numbers of metaphases with aberrations at any exposure duration and at any total culture time, irrespective of metabolic activation. The positive controls resulted in clear and statistically significant increases in metaphases with aberrations.

Gene mutation in mammalian cells

An in vitro mammalian cell gene mutation test according to OECD guideline 476 was performed withsorbitan monolaurate, ethoxylated dissolved in ethanol in mouse lymphoma L5178Y cells (Notox 2012). Cells were treated for 3 h without S9-mix at test substance concentrations of 0.3, 1, 3, 10, 33, 100 and 125 µg/mL and with 8% (v/v) S9-mix at 0.3, 1, 3, 10, 33, 100 and 300 µg/mL. Further, exposure for 24 h occured without S9-mix at 0.3, 3, 10, 33, 100, 150 and 190 µg/mL as well as with 12% (v/v) S9-mix at 0.3, 3, 10, 33, 100, 200, 300 and 350 µg/mL. Precipitation was observed at concentrations of 100 µg/mL and above. The mutation frequency was not increased in any of the treatment groups and no cytotoxicity was observed. Appropriate positive and negative controls were included in the experiment and revealed the expected results thereby validating the study.

Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
Genetic toxicity, in-vitro:
Gene mutation (Bacterial reverse mutation assay/Ames test): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A (according to OECD 471)
Chromosome aberration (in-vitro mammalian chromosome aberration test): negative with cultured human lymphocytes with and without metabolic activation (according to OECD 473).
Gene mutation (in-vitro mammalian cell gene mutation test): negative with cultured mouse lymphoma L5178Y cells with and without metabolic activation (according to OECD 476).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.